Abstract S100A1 and S100B are members of a multigenic family of Ca 2+-binding proteins of the EF-hand type highly abundant in astrocyte and striated muscle cells that have been implicated in the Ca 2+-dependent regulation of several intracellular activities including the assembly and disassembly of microtubules and type III intermediate filaments. In the present work we tested S100A1 and S100B for their ability to cause microtubule and/or intermediate filament disassembly in situ using triton-cytoskeletons obtained from U251 glioma cells and rat L6 myoblasts. Our results indicate that: (i) both proteins cause a Ca 2+-dependent disassembly of cytoplasmic microtubules in a dose-dependent manner; (ii) the S100A1- and S100B-inhibitory peptide, TRTK-12, blocks the S100A1 and S100B effects on microtubules; (iii) S100A1Δ88-93, an S100A1 mutant lacking the C-terminal extension, does not affect microtubule stability; and (iv) no obvious S100A1- or S100B-dependent intermediate filament disassembly could be observed under the experimental conditions used in the present study, but S100A1- and S100B-dependent microtubule disassembly results in a tendency of vimentin intermediate filaments to aggregate into bundles and/or to condense. Together, these results suggest that S100A1 and S100B probably cause microtubule disassembly by interacting with the microtubule wall, and that the two proteins do not affect intermediate filament stability via interaction with preformed intermediate filaments, in agreement with previous biochemical investigation. Our present data lend support to the possibility that S100A1 and S100B might have a role in the in vivo regulation of the state of assembly of microtubules in a Ca 2+-regulated manner and, potentially, on microtubule-based activities in astrocytes and myoblasts. Also, these data suggest that the both S100 proteins use their C-terminal extension for interacting with microtubules.